Collectin proteins are known to form stable oligomers. Collectins are one of 18 group members building the protein lectin superfamily containing a structural protein fold called C-type lectin domain (Zelensky et al., FEBS Journal 2005, Vol 272, p 6179-6217). Lectins are proteins that bind to carbohydrates and C-type lectins require calcium for binding. As the C-type lectin domain is involved in carbohydrate binding, this domain is also called the carbohydrate recognition domain (CRD). Collectins belong to the innate immunity and among other functions neutralize pathogens by binding to the carbohydrates e.g. present on viruses and bacteria. In addition, collectins regulate immune functions such as activation of complement and influencing inflammation. The basic structural features of collectins are a collageneous and a lectin domain which are the name giving components of collectins. Some members have been shown to contain additional structural features, thus they contain the following components: i) an N-terminal collagen domain connected to ii) an alpha-helical segment that is also referred to as the neck-region and iii) the CRD at the C-terminus (FIG. 35). Collectins trimerize non-covalently via the “triplehelical collagen”, the “coiled coil neck” and CRD regions. In humans, the collectin group contains serum mannose binding protein(s), collectins of the liver, kidneys, placenta and lung. Four lung collectins are known including pulmonary surfactant protein-A and -D (SP-A and SP-D) which contain N-terminal cysteines that are involved in the disulfide-mediatet oligomerization of pre-formed trimers. For instance, SP-D forms tetramers of trimers and SP-A forms hexamers of trimers (Kishore et al, Mol. Immunol. 2006, Vol. 43, 1293-1315)
In the attempt to provide trimeric complexes of TNF superfamily cytokines recombinant fusion proteins comprising a TNF cytokine and a multimerization component have been suggested as one possible approach (e.g. WO 0149866). The disclosed constructs however exhibited trimerization domains with a large molecular weight and with inefficient trimerization properties.
Schneider et al. (J Exp Med 187 (1989), 1205-1213) describes that trimers of TNF cytokines are stabilized by N-terminally positioned stabilization motifs. In CD95L, the stabilization of the CD95L-receptor binding domain trimer is presumably caused by N-terminal amino acid domains which are located near the cytoplasmic membrane.
Shiraishi et al. (Biochem Biophys Res Commun 322 (2004), 197-202) describes that the receptor binding domain of CD95L may be stabilized by N-terminally positioned artificial α-helical coiled-coil (leucine zipper) motifs. It was found, however, that the orientation of the polypeptide chains to each other, e.g. parallel or antiparallel orientation, can hardly be predicted. Further, the optimal number of hepta-d-repeats in the coiled-coil zipper motif are difficult to determine. In addition, coiled-coil structures have the tendency to form macromolecular aggregates after alteration of pH and/or ionic strength.
Mc Alinden et al. (J of Biol Chem, 2002, 277(43):41274-41281) discloses the preparation of a fusion protein between a human type IIA procollagen amino acid sequence and a 14 amino acid sequence corresponding to the first two heptad repeats of the rat surfactant protein's (SP-D) neck domain.
WO 01/42298 discloses the preparation of a fusion protein between surfactant protein-D comprising the signal sequence, the collagen domain and the neck domain and CD40L. The disadvantage of those fusion proteins is that they lead to multimeric aggregates that are highly immunogenic and that they do not produce biochemically defined trimeric ligands.
To circumvent the named problems existing in the art could be achieved by using collectin trimerization domains as a tool for forming controlled trimers. In the art attempts for this have been performed. However only the coiled-coil neck region of collectin trimerization domains (CRD) have been used in such attempts. The coiled-coil like neck-region of SP-D itself can be used as trimerisation domain, either N- or C-terminal fused to protein domains as described in WO95/31540.
However if using solely the coiled-coil neck region the optimal number of hepta-d-repeats to achieve a stable trimer (the overall length) are difficult to determine. The presented part of the SP-D neck region does not form sufficiently stable trimeres itself and needs to be optimized with respect to its length or repetition grade to generate stabilised trimeric fusion proteins. In addition, coiled-coil structures tend to form macromolecular aggregates after alteration of pH and/or ionic strength. Accordingly a collectin neck-region α-helical bundle exists only as a trimeric molecule in conditions which mimic or approximate physiological conditions. This implicates, that purification strategies employing pH-shifts and/or the alteration of the ionic strength might have a negative effect on the trimeric state of the neck solely based fusion proteins.
Also oligomerization of antibody fragments have been attempted by using collectin trimerization domains. E.g. fusion proteins comprising an anti-CD89-Fab or an anti-CD64-Fab fused to recombinant human fragment SP-D (neck+CRD-domain) have been investigated and found to be effective in targeting pathogens towards neutrophils. The fusion proteins presented had been generated by chemical crosslinking resulting in a mixture of protein products with the necessity of a complex purification regime to achieve the wanted protein species.
For human SP-D a mutant has been described in which amino acid phenylalanine 335 (corresponding to amino acid 355 of SEQ ID NO:21) has been mutated to alanine (SPD_F335A, Crouch et al., JBC 281: 18008-18014). This mutant showed very weak carbohydrate binding.
To allow for an efficient manufacturing process for Fab-SP-D based fusion proteins a process would be desirable that does not necessitate laborious purification procedures but allows for controlled production of defined products instead of crude mixtures.
The inventors found that the fusion proteins disclosed herein overcome the problems present in the art and allow for controlled generation of trimers of different effector polypeptides such as cytokines of the TNF superfamily or also of antibody fragments or single chain antibodies.
It was an object of the present invention to provide fusion proteins forming trimers which allow efficient recombinant manufacture combined with good trimerization properties and improved pharmaceutical properties.